Ultrashort Peptides as Bifunctional Nanomaterials

McCloskey, A., Gilmore, S., Porter, S., Gilmore, B., & Laverty, G. (2016). Ultrashort Peptides as BifunctionalNanomaterials. Poster session presented at PepMat 2016 , Barcelona, Spain.

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Download date:08. Oct. 2020

Results and discussion

The ability to self-assemble to form hydrogels is determined by the primary peptide structure. Naproxen and indomethacin conjugated peptides formed self-supporting hydrogels. Ibuprofen conjugates due to insufficient π-π stacking, remained asviscous solutions confirmed via viscosity measurements. Rheological analysis confirmed the hydrogels as viscoelastic withG’ being one order of magnitude higher than G’’ for all concentrations of peptides. FTIR further confirmed assembly withshoulders in the amide I region (1570 cm-1) and characteristic peaks representative of β-sheet formation (1625 cm-1) in anantiparallel arrangement (1675 cm-1). Higher ordered nanofibrous assemblies were observed under cryo-SEM and TEM.

The hydrogels proved to be relatively non-hemolytic and non-toxic in vitro. Strong antibiofilm activity was demonstrated by all hydrogels. The optimal reduction in percentage viable biofilm was exhibited by IndFFKK against Staphylococcus epidermidis. The anti-inflammatory activity of the NSAIDs was not inhibited following coupling to the peptide with hydrogels showing inhibitory activity against both COX-1 and COX-2 enzymes.

Ultrashort Peptides as Bifunctional Nanomaterials

McCloskey A.P, Gilmore S.M, Porter S.L, Gilmore B.F, Laverty G.School of Pharmacy, Queen’s University Belfast

IntroductionBiomaterials have contributed greatly to advances in modern medicine enhancing patients’ quality of life however they areregarded as foreign objects by the human body [1]. Their presence and sometimes trauma caused by insertion of abiomaterial triggers host inflammatory mediators. They also provide an ideal surface for bacterial attachment and biofilmformation. These can compromise biomaterial function and mechanical properties resulting in its failure or destruction ofwith associated negative effects on the patient and their outcomes [2].

Self-assembling ultrashort cationic peptides are an innovative form of antimicrobial hydrogels. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat pain and inflammation despite their systemic side effects. Acombination of these may create a novel self-assembling biomaterial with dual properties- a localized antimicrobial actionand ability to dampen the host immune response [3].

Gelation can be tailored to occur in response to physiological infective indicators: pH Enzymes Temperature

Thus facilitating targeted antimicrobial and anti-inflammatory activity at the site of infection [4]. This work focuses on thedevelopment of dual acting anti-inflammatory and antimicrobial self-assembling peptides with the incorporation of clinicallyused NSAIDs into our previously investigated self-assembling antimicrobial peptides with -FFKK.

References1. McCloskey, A. P., Gilmore, B. F., & Laverty, G. (2014). Evolution of Antimicrobial Peptides to Self-Assembled Peptides for Biomaterial Applications. Pathogens, 3(4), 791-821.2. Morais, J. M., Papadimitrakopoulos, F., & Burgess, D. J. (2010). Biomaterials/tissue interactions: possible solutions to overcome foreign body response. The AAPS journal, 12(2), 188-196.3. Li, J., Kuang, Y., Shi, J., Gao, Y., Zhou, J., & Xu, B. (2013). The conjugation of nonsteroidal anti-inflammatory drugs (NSAID) to small peptides for generating multifunctional supramolecular nanofibers/hydrogels. Beilstein journal of

organic chemistry, 9(1), 908-917. 4. Laverty, G., McCloskey, A. P., Gilmore, B. F., Jones, D. S., Zhou, J., & Xu, B. (2014). Ultrashort cationic naphthalene-derived self-Assembled peptides as antimicrobial nanomaterials. Biomacromolecules, 15(9), 3429-3439.

This investigation has shown that self-assembled NSAID conjugated peptide hydrogels may have potential clinically. Dual activity means that such molecules could act as novelcandidates for wound dressings and medical device coatings. Further to this, pH triggered assembly and cytotoxic investigations conducted thus far indicates the potential for targetedactivity at the necessary site of action. The next step will be to characterize further the biocompatibility and enzymatic stability of these molecules.

Fig.5. Percentage reduction of mature 24 h biofilms treated with 2%(w/v) NSAID conjugated hydrogels utilizing an alamarBlue assay. Resultsare displayed as a mean of 6 replicates.

Fig.4. 1-3: cryo-SEM and 4-6: TEM images of the NSAID peptide hydrogels at 2% (w/v).

Fig.6. Percentage inhibition of COX-1 and -2 enzymes following treatment with NSAID conjugated hydrogels. Results are displayed as a mean of 3 replicates.

Fig.1. Chemical structures of the NSAIDs conjugated to the synthesized peptides A) naproxen, B) ibuprofen, C) indomethacin.

A B C

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MethodsDiphenylalanine-dilysine peptides were synthesized on Wangresin following standard Fmoc protocols using a manualNitrogen bubbler apparatus. The final coupling step involvedaddition of one of the following NSAIDs (listed below) in thesame manner as to the addition of an amino acid:

naproxen (Npx) ibuprofen (Ibu) indomethacin (Ind)

Gelation was triggered by addition of sterile deionized water,raising the pH to ~pH 9 with 1M NaOH to ensure fulldissolution of the peptide. 0.5M HCl titrated the pH of thepeptide solution to near physiological pH (~pH 7.4). Gelationwas assessed after 24 hours via the inversion method.

The viscoelastic nature of the peptides was characterized byoscillatory rheology. Hydrogels with a storage modulus (G’)at least one order of magnitude greater than the lossmodulus (G’’) were classified as being viscoelastic.

Cryo-scanning electron microscopy (cryo-SEM) andtransmission electron microscopy (TEM) confirmed thepresence of nanofibrous networks. This was furtherconfirmed with evidence of beta stacking in anti-parallelarrangement determined by FTIR.

FTIR provided information regarding the secondary structure of the peptides. Hydrogels were prepared as detailed above but with deuterated solvents. Peaks were obtained from an average of 128 scans and wavelengths of 4000−400 cm-1.

Hemolytic activity was determined using equine erythrocytesand cytotoxicity against (NCTC929) murine fibroblast cells.Activity against cyclooxygenase -1 and -2 (COX-1 and -2)proved anti-inflammatory activity of the peptide hydrogels.

The antimicrobial properties of each concentration of peptidehydrogel were determined by the ability to reduce viable 24hour biofilms of both Gram-positive (Staphylococcus aureusATCC 6538 and Staphylococcus epidermidis ATCC 35984)and Gram-negative bacteria (Escherichia coli NCTC 11303and Pseudomonas aeruginosa PAO1).

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Fig.2. Frequency sweep and rheological properties for NSAIDconjugates at 2% (w/v). G’= storage modulus, G’’= loss modulus

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Fig.3. FTIR spectra for the NSAID conjugates at 2% (w/v).

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